Understanding the mechanism of controlled initiation of DNA replication remains a fundamental and largely unsolved problem in cell biology. Our long-term goal is to contribute to the solution of this problem by investigating Escherichia coli plasmid R6K (pR6K). The major focus of our research during the current granting period will be the biochemical analysis of the activator and replication inhibitor activities of pR6K- encoded initiator protein pi and the determination of the role of so- called enhancer region in replication. Because the gamma ori to which pi proteins binds shares a number of structural features with origins of many other plasmids, the proposed studies are likely to have broad biological significance. However, it is important to note that the gamma ori also differs from the other origins in many respects. Thus, our studies may elucidate novel mechanisms of control of DNA replication. Our specific aims in this proposal are: SA#1. To determine minimal sequence requirements for the activity of gamma ori replication in vivo. SA#2. To characterize in vitro the replication properties of gamma ori templates with and without the enhancer sequences. SA#3. To determine how the replication enhancer works. SA#4. To elucidate the role of pi protein in setting initiation frequency by using wt and mutant pi proteins.